(a) Field of the Invention
The invention relates to two different approaches using the Vpr/Vpx protein or p6 protein for treatment of HIV-1 and HIV-2 infections.
(b) Description of Prior Art
Acquired Immune Deficiency Syndrome (AIDS) is a slow degenerative disease of the immune and nervous systems caused by the Human Immunodeficiency Virus (HIV). The life cycle of HIV lies at the heart of the AIDS pandemic. The spread of the disease is primarily determined by the infectious properties of this virus. Progressive lethal degeneration of the immune and central nervous systems results from long term chronic replication of this virus.
HIV belongs to a unique virus family, the retroviridae, a group of small, enveloped, positive stranded, RNA viruses (Lavallee et al., 1994, J. Virol., 68:1926-1934; International Patent Application No. WO 90/158,75 on Dec. 27, 1990 in the name of DANA FARBER CANCER INSTITUTE). These viruses code for an enzyme, the reverse transcriptase (RT), which enables them to replicate their RNA genome through a DNA intermediate. Simple retroviruses contain three, contiguous reading frames coding for the gag, pol and env genes, which constitute their structural and enzymatic repertoire, all packaged in the progeny virion. The gag and env genes encode the core nucleocapsids proteins and the membrane glycoproteins of the virus, respectively, whereas the pol gene gives rise to the reverse transcriptase and other enzymatic activities (ribonuclease H, integrase and protease) that are essential for viral replication. HIV belongs to the lentivirus subfamily, members of which are characterized by several additional open reading frames (ORF) not found in simple retroviruses (FIG. 1). These ORFs all appear following gag-pol sequences, either immediately preceding the env sequences or overlapping it, and at least in one case, nef, extending well into the 3' Long Terminal Repeat (LTR). These ORFs code for non-structural viral proteins readily detectable in the cells. Much evidence has accumulated indicating that these gene products, collectively referred to as auxiliary proteins, are capable of modulating viral replication and infectivity.
HIV-1 possesses at least six such auxiliary proteins, namely, Vif, Vpr, Tat, Rev, Vpu and Nef. The closely related HIV-2 does not code for Vpu, but codes for another unrelated protein, Vpx, not found in HIV-1. Mutations affecting either Tat or Rev severely impair viral replication indicating that these two auxiliary proteins are essential for viral replication. However, at least in vitro, mutations affecting other auxiliary proteins result in minimal effect on the viral replication kinetics. Hence, these proteins have been dubbed dispensable or non-essential for in vitro replication, and are usually referred to as accessory gene products.
In the past few years, it has become evident that while these "accessory" genes are not required for productive replication, they are nonetheless capable of affecting replication events, even in vittro. More importantly, recent data indicates that they may affect pathogenesis in vivo.
The vpr gene encodes a 14 kDa protein (96 amino acids) in most strains of HIV-1 (FIG. 2; Myers et al., 1993, Human Retroviruses and AIDS 1993 I-II, Los Alamos National Laboratory, N.Mex., USA), although the open reading frame is often truncated in viral strains extensively passaged in tissue culture. The vpr open reading frame is also present in HIV-2 isolates and in most but not in all SIV isolates. A sequence similar to HIV-1 vpr is also found in Visna virus. The Vpr protein is made from a singly spliced rev-dependent mRNA species that accumulates late in infection. The Vpr protein of HIV and SIV have recently been shown to be present in mature viral particles in multiple copies. Amino acid comparison between vpx (a gene unique to HIV-2 and SIVs), and vpr from both HIV-2 and HIV-1, showed regions of strong homology (Tristem et al., 1992, EMBO J. 11: 3405-12). Like Vpr, Vpx is also packaged into the mature virion and has been shown to confer a growth advantage to viruses expressing the protein (Yu et al., 1993, J. Virol. 67: 4386-90). Interestingly, Vpx can be incorporated into HIV-1, HIV-2 or SIV with similar efficiencies. Based on the shared function, properties, including viral compartimentalization, and homologies of sequences between Vpr and Vpx, vpx in the HIV-2/SIV group, is thought to have arisen by duplication of the vpr gene (FIG. 2; Tristem et al., 1992, EMBO J. 11: 3405-12; and Myers et al., 1993, Human Retroviruses and AIDS 1993 I-II, Los Alamos National Laboratory, New Mexico, USA). Interestingly, Vpr and Vpx are the first regulatory protein of any retrovirus found to be associated with viral particles. Other regulatory proteins, such as tat, Rev, Nef, Vif and Vpu are not virion-associated. The assembly and maturation of HIV-1 viral particles is a complex process in which the structural Gag, Pol and Env gene products are expressed in the form of polyprotein precursors. The Gag proteins of HIV play a central role in virion assembly and budding. Gag proteins are initially synthesized as myristylated polyprotein precursors, Pr55.sup.gag and Pr160.sup.gag-pol, which are transported to the inner face of the plasma membrane where they can direct particle formation, even in the absence of other viral proteins. Complete budding leads to formation of immature particles, followed by HIV protease mediated cleavage of the Gag and Gag-Pol precursor polyproteins and formation of mature HIV particles with condensed core. The mature virion proteins derived from cleavage of the gag-encoded precursor, Pr55.sup.gag, include the p17 matrix protein (MA), the p24 capsid protein (CA), the p7 nucleocapsid protein (NC), and a small proline-rich peptide of approximately 6 kDa designated p6 which are linked in this order in the polyprotein precursor. Vpr is not part of the virus polyprotein precursors and its incorporation occurs by way of an interaction with a component normally found in the viral particle. It was recently reported that the HIV-1 Vpr could be incorporated in trans into viral-like particle (VLP) originating from expression of the Pr55.sup.gag only (Lavallee et al., 1994, J. Virol., 68:1926-1934). Data from this and other studies indicate that Vpr incorporation appeared to result from a direct interaction of Vpr with the carboxy-terminal region of the Pr55.sup.gag polyprotein (Paxton et al., 1993, J. Virol., 67(12):7229-7237; Lu et al., 1993, J. Virol., 67(1):6542-6550).
Functional studies indicated that the full length vpr protein could confer favorable growth properties to viruses. The increase in virion production is more pronounced in primary macrophages in both HIV-1 and HIV-2 systems, suggesting that Vpr function may be important in specific target cells. Interestingly, while mutations affecting HIV-1 vpr do not affect replication in peripheral blood mononuclear cells (PBMC), mutations in HIV-2 vpr results in a measurable impairment in these cells. Similarly, a recent study using anti-sense RNA directed against vpr inhibited viral replication in primary macrophages but not in transformed T-cells. Previous work indicated that this rapid growth advantage may be conferred by the weak transactivation property of Vpr on HIV-LTR directed gene expression (European Patent Application published under No. 474,797 on Mar. 18, 1992 in the name of DANA FARBER CANCER INSTITUTE). Cotransfection experiments suggest that vpr could augment the expression of a reporter gene from several heterologous promoters by approximately three to ten fold.
The carboxyl terminal sequence of Vpr have been shown to be important for Vpr mediated transactivation as prematurely truncated proteins are non-functional and are not packaged into the virion. Interestingly, a recent report also indicated that the carboxyl terminal of the protein is important for nuclear localization (Lu et al., 1993, J. Virol., 67(1):6542-6550). A specific vpr responsive LTR sequence was not identified and the exact mechanism by which vpr augments reporter gene expression is not clear. The precise mode of action of vpr is yet to be established. However, the presence of Vpr in the viral particle (a property also shared by Vpx) suggests that this protein has a role in the early stage of infection. Virion-associated non-structural proteins in many viral systems play a pivotal enzymatic functions in early replication steps, either because cellular homologues are unavailable or are sequestered, for example, in the nucleus. It is possible that Vpr is one such protein, capable of modulating early viral specific functions such as reverse transcription stabilization of early RNA or DNA intermediates, transport to the nucleus or integration. It is equally possible that Vpr could function at an early step, in a non-viral specific manner, by triggering processes that could make the cellular environment congenial to establish viral infection. In this regard, HIV-1 Vpr has been reported to be involved in inducing cellular differentiation in rhabdomyosarcoma cells (Levy et al., 1993, Cell, 72:541-550). Finally, because Vpr is synthesized late in the infection cycle of HIV, it may regulate the morphogenesis of the virus (late events) by an unknown mechanism or constitute a structural protein involved in the integrity of the virions.
The use of transport polypeptides for biological targeting is well known and was adapted to many fields. The HIV Tat protein has been described to effect the delivery of molecules into the cytoplasm and nuclei of cells (International Application published on Mar. 3, 1994 as No. WO 94/04686 in the name of BIOGEN, INC.). However, the Tat transport polypeptides can not allow the delivery of molecules to HIV virions. Viral proteins such as Gag of Rous sarcoma virus and Moloney murine leukemia virus and portion of HIV-1 Gag protein have been used as carrier for incorporation of foreign antigens and enzymatic markers into retroviral particles (Wang et al., 1994, Virology, 200:524-534). However, most of the Gag protein sequences are essential for efficient viral particles assembly, thus limiting the use of such virion components as carrier.
It would be highly desirable to be provided with means to target molecules to mature HIV-1 and HIV-2 virions to affect their structural organization and/or functional integrity.
It would also be highly desirable to be provided with a Vpr protein, a Vpx protein or fragments thereof which permit the development of chimeric molecules that can be specifically targeted into the mature HIV-1 and HIV-2 virions to affect their structural organization and/or functional integrity, thereby resulting in treatment of HIV-1 and HIV-2 infections.
It would also be highly desirable to be provided with a therapeutic agent which permit the targeting of chimeric molecules into the mature HIV-1 and HIV-2 virions as a treatment for HIV-1 and HIV-2 infections.
It would also be highly desirable to be provided with the identification of the protein interactions responsible for Vpr or Vpx incorporation into the mature HIV-1 and HIV-2 virions.
It would also be highly desirable to be provided with means to incorporate Vpr or Vpx into the mature HIV-1 and HIV-2 virions by making use of the protein interactions responsible for incorporation of Vpr or Vpx therein, thereby affecting the functional integrity of the HIV virions.
It would also be highly desirable to be provided with a Vpr protein fragment, a Vpx protein fragment, a p6 protein or p6 protein fragment which permits the development of molecules that can specifically interfere with the protein interactions responsible for Vpr or Vpx incorporation into the mature HIV-1 and HIV-2 virions to affect their functional integrity, thereby resulting in treatment of HIV-1 and HIV-2 infections.
It would also be highly desirable to be provided with a therapeutic agent which interferes with the protein interaction responsible for Vpr or Vpx incorporation in the mature HIV-1 and HIV-2 virions as a treatment for HIV-1 and HIV-2 infections.